Antimicrobial susceptibility testing is necessary to claim antibacterial activity of new chemicals. Euphorbia helioscopia has great medicinal importance because of its traditional uses and number of pharmacological activities. Various factors affect chemical composition of the plant which may ultimately modify its uses and activities. Antimicrobial susceptibility of Euphorbia helioscopia was tested by adopting CLSI, 2006; antimicrobial susceptibility testing guidelines. Agar well diffusion method and broth macrodilution method were used to validate antibacterial activity of standardized methanol extract and latex of E. helioscopia. Latex showed no antibacterial activity. E. coli (AI: 0.29 and MIC: 62.5 mg/mL), S. enterica (AI: 0.32 and MIC: 250 mg/mL), Staph. aureus (AI: 0.3and MIC; 250 mg/mL) showed susceptibility to leaves methanol extract.  The extract was found bactericidal against S. enterica as MIC and MBC were the same i.e. 250 mg/mL whereas, the extract showed relatively dose dependent activity against E. coli i.e.  bacteriostatic at 62.5 and 125 mg/mL and bactericidal at 250 mg/mL. However, extract showed bacteriostatic activity against Staph. aureus upto 250 mg/mL (highest dose employed).